Due to the existence of various contaminants in the cell culture medium, which include bacteria, yeast and fungi, different diagnostic methods have been considered. In this way, to detect bacteria from deformed cultured cells, detachment of adherent cells from the bottom of the container, movement of microscopic round components (round bacteria) or rod-shaped (rod-shaped bacteria), change the color of the environment to yellow color (usually the culture medium contains phenol rejection, which is added to the culture media as an indicator of pH change and gives the culture medium a pink color) and turbidity of the cell culture medium can be used. However, in fungal and yeast contamination, the presence of budding cell clumps or yeast, and the presence of wavy fungal colonies and filaments floating on the culture medium are essential for fungal contamination.

The ATCC (American Type Culture Collection), as a biological resource center, has performed cell line authentication and quality control tests on all cell lines. Therefore, obtaining information from different conditions of a cell line using the ATCC site is a reliable way to perform laboratory tests on a cell line. Thus, when publishing cell line research, the ATCC catalog number should be included in the research materials and methods section. Accordingly, the use of cell morphology using a microscope, cell growth curve analysis and cell doubling rate, cell line detection using isozyme electrophoresis, cell DNA testing or STR analysis (DNA fingerprinting) can be used in cell line detection.

There are several methods for assessing cell survival. Tests on the permeability of vital dyes through cell membranes are the easiest way to distinguish dead cells from living ones, usually with trypan blue dye. Other colors for this tests include Evans blue color, which indicates the degree of color permeability into the tissue, indicates cell damage and death, and lack of cell staining indicates cell survival. Calcine (Calcein AM), acridine orange and ethidium bromide are among the substances that show the survival rate of cells with health and DNA damage. These can be examined with a fluorescent microscope. Compounds that show survival in response to mitochondria with the formation of formazan deposits primarily contain materials that can be examined by spectrophotometry at a specific wavelength using MTT and XTT kits. Other materials that show mitochondrial membrane potential for cell survival and health include fluorescent dyes JC1, RHODAMIN 123, and some commercial kits that are tested by flow cytometry, and finally for assessing ROS or the extent of damaged DNA the tunnel technique can use. Also, flow cytometry indicates the survival or destruction of the cells.

In order to transfer the cell from outside the laboratory to the cell culture laboratory, the cell culture medium is pre-incubated to have sufficient CO2. The cell flask is then completely filled with this medium and can be placed in an incubator-free space for one day. In this case, the unfiltered flask is closed with a full lid and transferred at a temperature below 22 ° C in a suitable container in which the culture container is fixed and away from environmental contamination. For fresh tissue transfer (Fresh Tissue), if the tissue size is less than 0.5 x 0.5 cm, it can be about 3-5 hours in cell culture medium with HPSS buffer or Hank’s Balanced Salt. Solution transferred to prevent pH changes, or transferred with physiological serum. The transfer container should be done next to the dry ice and by completely covering the lid of the container with paraffin (to prevent contamination). It should be noted that for larger cell sizes in less than 1 hour, the sample should be transfer to the laboratory.

Cell culture media can be divided into two main categories: natural environments and synthetic culture media. Natural culture media are extracted from body secretions including plasma, lymph and serum or tissue extracts. Which are classified into three categories: Biological fluids, Tissue extracts and coagulants. In this category, FBS is the most well-known product that is added to synthetic culture media. The second category is synthetic culture media that are prepared using organic and inorganic compounds. Serum-containing media, Serum-free media, chemically defined media, Protein-free media and balanced salt solutions (BSS) are classified in this group. In all of these environments there are at least a few organic, inorganic compounds, non-essential amino acids, glutamines and vitamins. The most commonly used cultivation media include:

– MEM (Minimum Essential Medium)
– DMEM (Dulbecco’s – Modified Eagle’s Medium)
– IMDM (Iscove’s Modified Dulbecco’s Medium)
– RPMI-1640 & Ham’s F-10
These mediums are different in the amount compounds and therefore have different applications for different cell lines.

Freezing is a well-known laboratory method for storing cells and other biological substances in liquid nitrogen at temperatures close to -196 ° C. For this purpose, before performing the cell freezing steps, it is necessary to first check the survival rate of cells with the vital dye trypan blue. On the other hand, the presence of Mycoplasma cell infection should be determined before cell freezing. In addition, one or two days before freezing, the cells should be passage and fresh medium should be poured on the cells to allow the cells to multiply. Cells with a density of 70 to 80% are usually frozen in a suitable culture dish and 500,000 to one million cells are poured into each cryotubes. The cell freeze medium typically contains culture medium, an antifreeze such as DMSO or glycerol, and a protein source such as FBS serum. One milliliter of freezer material is combined with one million cells and placed overnight at a freezing rate of 1 ° C / minute to -80 °C for freezing. Then, it is placed in liquid nitrogen vapor and finally in liquid nitrogen. In order to defrost the cell, the cryovial is removed from the nitrogen tank at 37 ° C to melt the contents. The preheated culture medium is then immediately added to these contents up to three times its volume, and after centrifugation at 1500 rpm, the ambient temperature is removed for 5 minutes, the previous medium and the fresh medium are added to the cell and the cell is cultured.